Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. The disclosure of these references are hereby incorporated by reference into the present disclosure.
Condensed tannins (also called proanthocyanidins) are plant phenolic compounds which are structurally related to the anthocyanins that cause pumple and red colours in flowers. Specifically, condensed tannins are 1,4-linked and 1,6-linked polymers of flavan-4-ols, derived by condensation from several products of the phenylpropanoid/flavonoid pathway (Table 1) (Gruber et al., 1999; Peterson et al., 1999). The biosynthesis of these two classes of compounds, i.e., tannins and anthocyanins, occurs in plants using a set of common genes, after which the pathway diverges and unique genes are required for each class. Many plant species accumulate condensed tannins in their vegetative, floral and seed tissues (Porter 1988). Legumnes are a particularly rich source of these compounds. The legumes sainfoin (Onobychis viciifolia) and big trefoil (Lotus uliginosus) contain substantial levels of condensed tannins in leaf and other vegetative tissue and in seed coats. With the exception of barley and sorghum seedcoats (Butler 1982; Erdal 1986) and one report in rice (Reddy et al., 1995), the major cereal crops do not express condensed tannins. Several other species such as alfalfa, white clover, L. japonicus and the oilseed Brassica, only express condensed tannins in seedcoats.
The biological properties of tannins are related to their chemical structure. Their polymeric phenol nature facilitates hydrogen bonding with proteins in preference to other molecules (Hagerman and Butler 1981). The combination of hydroxyl groups (which can easily ionize to form quinone) with the ortho position of hydroxyl groups on ring B (which facilitates metal binding), contribute to their antioxidant properties and their ability to protect from excess sunlight.
Alfalfa (luceme; Medicago sativa or M. falcata) produces a linear procyanidin (3′4′-OH) condensed tannin polymer in the testa layer of the seedcoat as well as several smaller secreted flavonoids, while the leaves normally produce flavone glycosides instead of tannins (Koupai et al., 1993; Olah and Sherwood, 1971; Saleh et al., 1982). Chalcone synthase (CHS) and dihydroflavonol reductase (DFR) are inconsistently expressed in alfalfa leaves, while the flavanone 3B-hydroxylase (F3H) gene is not detected at all in alfalfa leaves (Charrier et al., 1995; Junghans et al., 1993; Skadauge et al., 1997; Ray and Gruber unpublished).
Leucoanthocyanidin reductases (LARs) comprise the first step committed exclusively to condensed tannins in the flavonoid pathway. LARs are normally expressed only in tannin-containing tissue (Skadhauge, 1996; Koupai-Abyazani et al., 1993; Singh et al., 1997; Joseph et al., 1998). In alfalfa, LCR (3′4′-OH-specific LAR) activity is high only during early seed development, but cannot be detected in leaves (Skadhauge et al., 1997). The flavanone 3B-hydroxylase gene (F3H) and 3′4′-OH-specific leucoanthocyandin reductase gene (LCR) are two functional blocks that prevent alfalfa leaves from accumulating condensed tannins.
Natural and induced mutants affecting condensed tannin or anthocyanin expression have been identified in various crop and forage plant species, including sorghum, barley, pea, Arabidopsis, rice and Lotus japonicus (Butler et al. 1982; Gruber et al. 1996; Jende-Strid 1993; Koorneef et al. 1982; Koorneef 1991; Jambunathan et al.1986; Reddy et al. 1995). However, no mutations or variants with leaf tannin have been found in alfalfa or related Medicago species (Goplen et al., 1980). A somaclonal variant of alfalfa with a small but detectable content of leaf bud flavan-3-ol was recovered (Lees et al., 1992), but tannin could not be extracted from the buds and the trait proved unstable. Somatic hybridization between sainfoin and alfalfa has been used to develop alfalfa-like hybrids with sainfoin DNA, but to date no plants have been recovered with stable leaf tannin contents (Larkin et al., 1998). Alfalfa only accumulates anthocyanins in senescing leaves.
Some forage legume species express condensed tannins in leaves and other vegetative tissues. These include sainfoin, big trefoil (L. uliginosus), L. angustissimus, all of which express high levels of leaf condensed tannins. Birdsfoot trefoil (L. corniculatus) expresses leaf condensed tannin at a moderate level, while the related L. japonicus does not express leaf condensed tannin. All of these express condensed tannin in seed coat (Gruber et al., 1999).
The alteration of various intermediates in the phenylpropanoid/flavonoid pathway in certain plants has been demonstrated or suggested to be advantageous for certain uses. For example, certain flavonoids have been suggested to have the ability to inhibit phytopathogens in certain plant species. Flavonoid levels have been manipulated in order to select particular flower colours and patterns. Moreover, increased amounts of condensed tannins in certain forage crops have been found to be useful for decreasing bloat in cattle, improving ruminal protein bypass, reducing intestinal parasites, and reducing sileage degradation by proteolysis.
Researchers have attempted to alter the flavonoid pathway in order to manipulate condensed tannin synthesis in certain plants. Variations in the ability to affect changes in anthocyanin and condensed tannin expression have been observed using the maize C1 gene (myb-like) and myc-like genes constitutively expressed in maize, Arabidopsis, chrysanthemum, tomato, petunia, and oats (Lloyd, 1992; Cone et al., 1986; Paz-Arez et al., 1987; Wong et al., 1991; Bradley et al., 1998). For example, the combination of B-Peru (myc-like), and C1 induced anthocyanin production in wheat, barley and oats (Wong et al., 1991). The combination of B-Peru and C1 increased anthocyanin expression only slightly in white clover (maximum 2% of expression level in maize) and in peas (maximum 20% of expression level in maize, except for petal tissue) (de Majnik et al., 1998). B-Perm and C1 were expressed in Arabidopsis, and stimulated anthocyanin production in leaves (Lloyd et al., 1992). Lc (myc-like) stimulated anthocyanin expression in Brassica napus (Babwah et al., 1998), and in petunia (Bradley et al., 1998), but not in pelargonium or lisianthus (Bradley et al., 1999). A related maize anthocyanin regulatory gene, Sn (myc-like) has been introduced into birdsfoot trefoil (Lotus conriculatus) and caused hairy root cultures to become pigmented (Damiani et al., 1998). Unexpectedly, condensed tannins and tannin genes, which are normally elevated in leaves of Lotus corniculatus, were either completely suppressed or unaffected in transgenic plants with the Sn gene, while root tannin levels were elevated (Damiani et al., 1999). These authors have recently been able to raise leaf levels with Sn (Damiani, personal communication).
PCT/AU97/00529 is directed to nucleic acids and their encoded polypeptides involved in condensed tannin biosynthesis and their use in regulating the biosynthesis and accumulation of condensed tannins in plants. The nucleic acids are believed to encode leucoanthocyanidin reductases (Lar) of plants.
PCT/GB93/00019 is directed to a method for regulating the expression of one or more anthocyanin pigment genes in a plant. PCT/CA99/00056 is directed to methods and compositions for the alteration of compounds produced by secondary metabolic pathways in plants. Canadian patent application 2,130,800 is directed to a nucleotide sequence encoding flavonoid-3′,5′-hydroxylase activity to alter pigment patterns in a transformed plant. PCT/EP99/00419 is directed to the use of certain transcription factor genes for flavonoid biosynthesis in order to manipulate the production of flavonoids other than anthocyains in plants. WO 99/09810 is directed to alfalfa plants having measurable endogenous tannin levels for use as alfalfa forage for improved ruminant health and nutrition and methods of identifying and breeding tannin-expressing alfalfa plants.
Identification of genes which regulate the synthesis of condensed tannins in plants, or of genes regulating the supply of substrate for the condensed tannin branch of the flavonoid pathway may provide a means of developing methods to manipulate the tannin levels of plants advantageously. Such genes and methods could be used, for example, to develop alfalfa with leaves containing moderate condensed tannin levels for improved forage quality, as well as for the development of condensed tannins in canola vegetative tissues to provide insect resistance.
The complexity of the phenylpropanoid/flavonoid pathway often makes it difficult to successfully target specific compounds in the pathway using transgenic constructs and methods to generate stably transformed plants. This is often the case with the prior art. In the present application, the Applicant demonstrates the transformation of alfa with Lc, a regulatory gene of the basic helix-helix-loop or myc class. A spectrum of transgenic plants, ranging from no colour change to plants with dark red/green leaf and stem colouration indicative of anthocyanin accumulation, was obtained using a construct containing the Lc sequence. These results indicate that regulatory genes of this class can stimulate alfalfa leaf flavonoid genes to synthesize substrates all the way down to the branchpoint leading to either anthocyanins or condensed tannins. Thus stably transformed alfalfa plants can be made with improved characteristics for use. Lc has not previously been used to transform alfalfa or other forage legumes as such transformation is not a simple straightforward process.